Circular Muscle Research Articles

Altered Esophageal Smooth Muscle Phenotype in Achalasia.

Achalasia is a disorder characterized by impairment in lower esophageal sphincter relaxation and esophageal aperistalsis, caused primarily by loss of inhibitory innervation. However, little is known about associated changes in esophageal smooth muscle. We examined the contractile phenotype and innervation of the circular smooth muscle, as well as inflammatory status, and correlated these with patient-specific parameters. Circular smooth muscle biopsies were obtained in consecutive patients with achalasia undergoing peroral endoscopic myotomy. Axonal innervation and neurotransmitter subtypes were determined with immunocytochemistry, and this was used with quantitative Polymerase Chain Reaction (qPCR) to characterize smooth muscle proliferation and cellular phenotype, as well as collagen expression. These were compared to control tissue obtained at esophagectomy and correlated with patient demographic factors including age, onset of symptoms, and Eckhardt score. Biopsies of smooth muscle were obtained from 25 patients with achalasia. Overall, there was increased mast cell number and collagen deposition but increased smooth muscle cell proliferation vs control. There was a striking drop in axon density over controls, with no differences among subtypes of achalasia. Immunocytochemical analysis showed increased expression of the contractile marker α-smooth muscle actin, principally in Type 1 achalasia, that increased with disease duration, while qPCR identified increased mRNA for smoothelin with decreased myosin heavy chain and collagen 3a1, but not collagen 1a1. The thickened circular smooth muscle layer in achalasia is largely denervated, with an altered contractile phenotype and fibrosis. Biopsies obtained during peroral endoscopic myotomy provide a means to further study the pathophysiology of achalasia.

The Protective Effect of Lithium Against Rotenone may be Evolutionarily Conserved: Evidence from Eisenia fetida, a Primitive Animal with a Ganglionic Brain.

Chronic exposure to stress is a non-adaptive situation that is associated with mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), especially superoxide anion (SA). This accumulation of ROS produces damage-associated molecular patterns (DAMPs), which activate chronic inflammatory states and behavioral changes found in several mood disorders. In a previous study, we observed that an imbalance of SA triggered by rotenone (Ro) exposure caused evolutionarily conserved oxi-inflammatory disturbances and behavioral changes in Eisenia fetida earthworms. These results supported our hypothesis that SA imbalance triggered by Ro exposure could be attenuated by lithium carbonate (LC), which has anti-inflammatory properties. The initial protocol exposed earthworms to Ro (30 nM) and four different LC concentrations. LC at a concentration of 12.85mg/L decreased SA and nitric oxide (NO) levels and was chosen to perform complementary assays: (1) neuromuscular damage evaluated by optical and scanning electron microscopy (SEM), (2) innate immune inefficiency by analysis of Eisenia spp. extracellular neutrophil traps (eNETs), and (3) behavioral changes. Gene expression was also evaluated involving mitochondrial (COII, ND1), inflammatory (EaTLR, AMP), and neuronal transmission (nAchR α5). LC attenuated the high melanized deposits in the circular musculature, fiber disarrangement, destruction of secretory glands, immune inefficiency, and impulsive behavior pattern triggered by Ro exposure. However, the effects of LC and Ro on gene expression were more heterogeneous. In summary, SA imbalance, potentially associated with mitochondrial dysfunction, appears to be an evolutionary component triggering oxidative, inflammatory, and behavioral changes observed in psychiatric disorders that are inhibited by LC exposure.

Omnidirectional compliance on cross-linked actuator coordination enables simultaneous multi-functions of soft modular robots

Earthworms have entirely soft bodies mainly composed of circular and longitudinal muscle bundles but can handle the complexity of unstructured environments with exceptional multifunctionality. Soft robots are naturally appropriate for mimicking soft animal structures thanks to their inherent compliance. Here, we explore the new possibility of using this compliance to coordinate the actuation movements of single-type soft actuators for not only high adaptability but the simultaneous multifunctionality of soft robots. A cross-linked actuator coordination mechanism is proposed and explained with a novel conceptual design of a cross-linked network, characterization of modular coordinated kinematics, and a modular control strategy for multiple functions. We model and analyze the motion patterns for these functions, including grabbing, manipulation, and locomotion. This further enables the combination of simultaneous multi-functions with this very simple actuator network structure. In this way, a soft modular robot is developed with demonstrations of a novel continuous-transportation mode, for which multiple objects could be simultaneously transported in unstructured environments with either mobile manipulation or pick-and-place operation. A comprehensive workflow is presented to elaborate the cross-linked actuator coordination concept, analytical modeling, modular control strategy, experimental validation, and multi-functional applications. Our understanding of actuator coordination inspires new soft robotic designs for wider robotic applications.

To Compare the Effectiveness of Kegel’s Exercise and Paula Method in women with Urinary Incontinence

Objective of The Study: The study aims to compare the effectiveness of Paula method and Kegel's exercise in women with urinary incontinence. Background of The Study: Pelvic floor muscle weakness is one of the cause for urinary incontinence. Conservative management such as pelvic floor muscle training and Paula method is commonly recommended as first-line therapy for women with urinary incontinence. The purpose of the study is to examine the efficacy of circular muscle strengthening (Paula method) as opposed to pelvic floor muscle training (PMFT) on stress urinary incontinence. Methodology: This study was a comparative study design with pre and post type with 40 subjects randomly divided into two groups (group A and group B). Group A received Kegel’s exercise and Group B received Paula method. Result: On comparing Pre test and Post test within Group A and Group B of ICIQ score, QOL score, sexuality questionnaire score shows highly significant difference in mean values at P ≤ 0.001. Conclusion: This study concludes that Paula method decreases frequency of urinary incontinence by strengthening sphincter muscles. Compared to the Kegel’s exercise, Paula method showed greater difference in decreasing the frequency of urinary incontinence.

Plasticity of colonic enteric nervous system following spinal cord injury in male and female rats.

Neurogenic bowel is a dysmotility disorder following spinal cord injury (SCI) that negatively impacts quality of life, social integration, and physical health. Colonic transit is directly modulated by the enteric nervous system. Interstitial Cells of Cajal (ICC) distributed throughout the small intestine and colon serve as specialized pacemaker cells, generating rhythmic electrical slow waves within intestinal smooth muscle, or serve as an interface between smooth muscle cells and enteric motor neurons of the myenteric plexus. Interstitial Cells of Cajal loss has been reported for other preclinical models of dysmotility, and our previous experimental SCI study provided evidence of reduced excitatory and inhibitory enteric neuronal count and smooth muscle neural control. Immunohistochemistry for the ICC-specific marker c-Kit was utilized to examine neuromuscular remodeling of the distal colon in male and female rats with experimental SCI. Myenteric plexus ICC (ICC-MP) exhibited increased cell counts 3 days following SCI in male rats, but did not significantly increase in females until 3 weeks after SCI. On average, ICC-MP total primary arborization length increased significantly in male rats at 3-day, 3-week, and 6-week time points, whereas in females, this increase occurred most frequently at 6 weeks post-SCI. Conversely, circular muscle ICC (ICC-CM) did not demonstrate post-SCI changes. These data demonstrate resiliency of the ICC-MP in neurogenic bowel following SCI, unlike seen in other related disease states. This plasticity underscores the need to further understand neuromuscular changes driving colonic dysmotility after SCI in order to advance therapeutic targets for neurogenic bowel treatment.

Characterization of rat gastric myogenic contractions and modulation by oxytocin and arginine-vasopressin

BackgroundInterstitial cells of Cajal generate slow wave gastric electrical activity, initiating spontaneous muscle contractions. This becomes dysrhythmic during nausea when [Arg8]-vasopressin (AVP) is also released. In human stomach AVP increased spontaneous contraction activity and muscle tone, not neuronally-mediated contractions. Rodents cannot vomit, releasing the related hormone, oxytocin (OT) instead. We hypothesised that rat stomach would behave differently. Experimental approachSpontaneous and electrically-evoked (EFS) contractions were measured in rat forestomach and antrum circular muscle. Custom software defined spontaneous contractions by analysing eight motility parameters. ResultsThe forestomach was quiescent. Irregular antrum contractions became regular adjacent to the pylorus (1.7 ± 0.4 mN; 1.2 ± 0.1 contractions/min, n = 12). These were unaffected by tetrodotoxin (10−6 M), atropine (10−6 M) and L-NAME (3 × 10−4 M). In both regions, AVP (pEC50∼9.0) and OT (∼0.5 log10-unit less potent) caused contraction (greater in antrum), competitively antagonized by, respectively, SR49059 (pKB∼9.5) and L371257 (pKB∼9.0), reduced by tetrodotoxin but unaffected by atropine. In the antrum, AVP and OT (∼2 log10-units less potent/efficacious) regularized and increased spontaneous contraction amplitude, frequency, rates of contraction/decay. In both regions, EFS-evoked contractions, abolished by atropine/tetrodotoxin, were reduced by AVP and OT, with AVP more potent and efficacious, particularly in forestomach. ConclusionIrregular spontaneous contractions of gastric antrum suggest variable ICC-muscle coupling. AVP and less potently, OT, enhanced frequency and force of contractions via V1A and OT receptors. Compared with human, differences in contraction regularity, potency and ability of AVP/OT to affect neuronal function suggests caution when using rat stomach to model ICC functions and nauseagenic stimuli.

Interstitial cells of Cajal in the ureteropelvic junction: does its expression have a role in the pathogenesis of congenital ureteropelvic junction obstruction?

Background One of the most frequent urological abnormalities in pediatric urology and the most frequent cause of hydronephrosis in the pediatric age group is uteropelvic <ureteropelvic?> junction obstruction (UPJO). Although its etiology has been the subject of several investigations, it is still unknown. Objective Comparing the quantitative changes in the interstitial cells of Cajal (ICC) between specimens with UPJO and specimens without hydronephrosis using light microscopy and correlating these changes to the etiology of the disease. Methods Patients with UPJO and patients without obstruction were compared immunohistochemically with c-kit (CD117) between February 2020 and February 2022 at Ain Shams University Hospital to quantify the ICC, which is situated close to the circular muscle layer and parallel to the muscle cells. Results Light microscopic analysis revealed that the control group’s Cajal cell density was much higher than the number of cells in the patients with UPJ blockage. Conclusion Our research found that the mean density of the ICC was lower in UPJO patients compared with controls, indicating that the absence of the ICC may contribute to the pathogenesis of UPJO.

The In Vitro Contractile Response of Canine Pregnant Myometrium to Oxytocin and Denaverine Hydrochloride.

In pregnant bitches, the response to oxytocin and denaverine hydrochloride in dystocia management is usually poor. To better understand the effect of both drugs on myometrial contractility, the circular and longitudinal muscle layers were examined in an organ bath. For each layer, three myometrial strips were stimulated twice, each with one of three oxytocin concentrations. The effect of denaverine hydrochloride was studied once in direct combination with oxytocin and alone with subsequent oxytocin administration. Contractions were recorded and evaluated for average amplitude, mean force, area under the curve (AUC), and frequency. Effects of different treatments were analyzed and compared within and between layers. In the circular layer, oxytocin significantly increased amplitude and mean force compared to untreated controls regardless of stimulation cycles or concentrations. In both layers, high oxytocin concentrations caused tonic contractions, while the lowest concentration created regular rhythmic contractions. Longitudinal layer tissue responded to oxytocin with a significantly decreased contractility when stimulated twice, presumably a sign of desensitization. Denaverine hydrochloride neither affected oxytocin induced contractions nor showed a priming effect to subsequent oxytocin. Thus, no benefit of denaverine hydrochloride on myometrial contractility was found in the organ bath. Our results suggest a better efficiency of low-dose oxytocin in canine dystocia management.

Diagnostic methods to measure spastic segment and guide tailored myotomy length in type 3 achalasia.

Myotomy length in type 3 achalasia is generally tailored based on segment of spasticity on high-resolution manometry (HRM). Potential of length of tertiary contractions on barium esophagram (BE) or length of thickened circular muscle on endoscopic ultrasound (EUS) to guide tailored myotomy is less understood. This study aimed to assess agreement between spastic segments lengths on HRM, BE, and EUS among patients with type 3 achalasia. This retrospective study included adults with type 3 achalasia on HRM between November 2019 and August 2022 who underwent evaluation with EUS and/or BE. Spastic segments were defined as HRM-distance between proximal borders of lower esophageal sphincter and high-pressure area (isobaric contour ≥70 mmHg); EUS-length of thickened circular muscle (≥1.2 mm) from proximal border of esophagogastric junction (EGJ) to the transition to a non-thickened circular muscle; BE-distance between EGJ to proximal border of tertiary contractions. Pairwise comparisons assessed for correlation (Pearson's) and intraclass correlation classification (ICC) agreement. Twenty-six patients were included: mean age 66.9 years (SD 13.8), 15 (57.7%) male. Spastic segments were positively correlated on HRM and BE with good agreement (ICC 0.751, [95% CI 0.51, 0.88]). Spastic segments were negatively correlated with poor agreement on HRM and EUS (ICC -0.04, [-0.45, 0.39]) as well as BE and EUS (ICC -0.03, [-0.47, 0.42]). Length of spastic segment was positively correlated on HRM and BE while negatively correlated when compared to EUS, supporting the common use of HRM and highlighting the uncertain role for EUS in tailoring myotomy length for type 3 achalasia.

Abnormally elevated expression of ACTA2 of circular smooth muscle leads to hyperactive contraction in aganglionic segments of HSCR

BackgroundActin Alpha 2 (ACTA2) is expressed in intestinal smooth muscle cells (iSMCs) and is associated with contractility. Hirschsprung disease (HSCR), one of the most common digested tract malformations, shows peristaltic dysfunction and spasm smooth muscles. The arrangement of the circular and longitudinal smooth muscle (SM) of the aganglionic segments is disorganized. Does ACTA2, as a marker of iSMCs, exhibit abnormal expression in aganglionic segments? Does the ACTA2 expression level affect the contraction function of iSMCs? What are the spatiotemporal expression trends of ACTA2 during different developmental stages of the colon?MethodsImmunohistochemical staining was used to detect the expression of ACTA2 in iSMCs of children with HSCR and Ednrb−/− mice, and the small interfering RNAs (siRNAs) knockdown technique was employed to investigate how Acta2 affected the systolic function of iSMCs. Additionally, Ednrb−/− mice were used to explore the changes in the expression level of iSMCs ACTA2 at different developmental stages.ResultsThe expression of ACTA2 is higher in circular SM in the aganglionic segments of HSCR patients and Ednrb−/− mice than in normal control children and mice. Down regulation of Acta2 weakens the contraction ability of intestinal smooth muscle cells. Abnormally elevated expression of ACTA2 of circular smooth muscle occurs since embryonic day 15.5 (E15.5d) in aganglionic segments of Ednrb−/− mice.ConclusionsAbnormally elevated expression of ACTA2 in the circular SM leads to hyperactive contraction, which may cause the spasm of aganglionic segments in HSCR.

Retracted: PKC-delta and PKD activate MAPK signal pathway in mechano-transcription of colonic smooth muscle cells.

Retraction: [PKC-delta and PKD activate MAPK signal pathway in mechano-transcription of colonic smooth muscle cells, Z. Yang, K. He, T. Wang, et al. Neurogastroenterology & Motility 2023; e14623 (https://onlinelibrary.wiley.com/doi/full/10.1111/nmo.14623)]. The above article, published online on June 6, 2023 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the Journal Editor in Chief, Maura Corsetti, and John Wiley & Sons Ltd. The retraction has been agreed due to unat[1]tributed overlap between this article and the abstract published in Gastroenterology: Li F, Sarna SK and Shi XP. Roles of PKCs and PKD in Mechanotranscription in Colonic Smooth Muscle Cells: Inhibition of Mechanotranscription as a Potential Treatment for Motility Dysfunction in Obstructive Disorders. In: 2012 Digestive Disease Week Abstract Supplement; May 19-22, San Diego, CA. Abstract 120 (https://www.gastrojournal.org/article/S0016-5085(12)60115-2/pdf).

Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells.

The distribution, morphology, and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia, and macrophages are far from being complete. The vessels in the adult mouse colon were stained by the cardiovascular perfusion of wheat germ agglutinin (WGA)-Alexa Fluor 448 and by CD31 immunoreactivity. Nerve fibers, enteric glia, and macrophages were immunostained in the WGA-perfused colon. The blood vessels entered from the mesentery to the submucosa and branched into the capillary networks in the mucosa and muscularis externa. The capillary net formed anastomosed rings at the orifices of mucosa crypts, and the capillary rings surrounded the crypts individually in the proximal colon and more than two crypts in the distal colon. Microvessels in the muscularis externa with myenteric plexus were less dense than in the mucosa and formed loops. In the circular smooth muscle layer, microvessels were distributed in the proximal, but not the distal colon. Capillaries did not enter the enteric ganglia. There were no significant differences in microvascular volume per tissue volume between the proximal and distal colon either in the mucosa or muscularis externa containing the myenteric plexus. PGP9.5-, tyrosine hydroxylase-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers were distributed along the vessels in the submucosa. In the mucosa, PGP9.5-, CGRP-, and vasoactive intestinal peptide (VIP)-immunoreactive nerves terminated close to the capillary rings, while cells and processes labeled by S100B and glial fibrillary acidic protein were distributed mainly in the lamina propria and lower portion of the mucosa. Dense Iba1 immunoreactive macrophages were closely adjacent to the mucosal capillary rings. There were a few macrophages, but no glia in apposition to microvessels in the submucosa and muscularis externa. In conclusion, in the mouse colon, (1) the differences in vasculature between the proximal and distal colon were associated with the morphology, but not the microvascular amount per tissue volume in the mucosa and muscle layers; (2) the colonic mucosa contained significantly more microvessels than the muscularis externa; and (3) there were more CGRP and VIP nerve fibers found close to microvessels in the mucosa and submucosa than in the muscle layers.

Induction of apoptosis, oxidative stress, hormonal, and histological alterations in the reproductive system of thiamethoxam-exposed female rats

The present study aimed to investigate the oral toxic effects of 1/10 LD50 and 1/5 LD50 of thiamethoxam (TMX), a neonicotinoid insecticide, on the reproductive system of female Wistar rats. Thirty female rats were divided into three groups and supplied orally with either; saline solution, 1/10 LD50 of TMX (156 mg/kg) or 1/5 LD50 of TMX (312 mg/kg). The daily administration was extended for 30 days. Investigating the parameters of oxidative stress, hormonal levels, histopathological alterations, and the apoptotic markers (P53, BAX, BCL-2, and caspase-3) was performed in the uterus and ovary of rats. Results showed significant changes in the body weight gain, and relative weight of the left and right ovaries and uterus. Moreover, luteinizing hormone (LH), estradiol (ED), and progesterone (PG) serum levels were not significantly altered following TMX oral administration. The level of follicle-stimulating hormone in the TMX-exposed group (156 mg/kg) was significantly increased; however, a significant decrease was observed in TMX-exposed animals (312 mg/kg). TMX induced significant oxidative stress in exposed groups by reducing the activities of superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT), and elevating malondialdehyde (MDA) levels. Following hematoxylin and eosin staining, the microscopic examination revealed deteriorated luteal cells with vacuolation in the corpus luteum, a follicle containing a degenerated oocyte and degeneration/necrosis of the circular muscle layer with a high rate of apoptotic cells in TMX-exposed animals. TMX induced transcriptional alterations in apoptosis-related genes shifting towards the activation of the intrinsic apoptotic pathway. Collectively, results suggest the toxic effect of the TMX on the reproductive health of female Wistar rats.

Diagnostic challenges of hypoganglionosis based on immunohistochemical method.

Hypoganglionosis resembles Hirschsprung's disease as in both diseases, patients may present with severe constipation or pseudo-obstruction. To date, diagnosis of hypoganglionosis is still difficult to be established due to lack of international consensus regarding diagnostic criteria. This study aims to evaluate the use of immunohistochemistry to provide objective support for our initial subjective impression of hypoganglionosis as well as to describe the morphological features of this study. This is a cross-sectional study. Three resected intestinal samples from patients with hypoganglionosis at Kyushu University Hospital, Fukuoka, Japan were included in this study. One healthy intestinal sample was used as control. All specimens were immunohistochemically stained with anti-S-100 protein, anti-α-smooth muscle actin (α-SMA), and anti-c-kit protein antibodies. (I) S-100 immunostaining: hypoplasia of the myenteric ganglia and marked reduction of intramuscular nerve fibers were observed in several segments of the intestine. (II) α-SMA immunostaining: the pattern of the muscular layers was almost normal in all segments; however, some areas showed hypotrophy of the circular muscle (CM) layers and hypertrophy of the longitudinal muscle (LM) layers. (III) C-kit immunostaining: a decreased in the number of interstitial cells of Cajal (ICCs) was observed in almost all segments of the resected intestine, even around the myenteric plexus. Each segment of intestine in hypoganglionosis had different numbers of ICCs, sizes, and distributions of ganglions, as well as patterns of musculature, which may range from severely abnormal to nearly normal. Further investigations regarding the definition, etiology, diagnosis, and treatment of this disease should be performed to improve the prognosis of this disease.

Identification and characterization of Myxobilatus channi n. sp. (Cnidaria: Myxosporea) infecting the alimentary tract of northern snakehead Channa argus cultivated in China

The northern snakehead Channa argus is a freshwater fish of high economic importance in Asian countries. Intensive snakehead cultivation requires the regular addition of feedwater to ensure water quality, providing an opportunity for parasite transmission. However, the parasite composition of northern snakehead is still poorly studied. In a series of investigations conducted in China, a myxosporean species was isolated from the farmed northern snakehead. It manifested with a dense formation of ellipsoidal, proliferous cysts presenting along the wall of the intestine and pyloric caeca. The microorganism inside the cyst displayed elongate, pyriform, and bivalved myxospores, with an elongated caudal appendage extending from posterior extremity of each smooth shell valve. Two pyriform polar capsules were situated at a level perpendicular to the sutural plane. Based on these observations, the present isolates was assigned to the genus Myxobilatus. However, it was morphologically distinguishable from other congeners. Moreover, the same result was obtained according to a less than 92% top genetic identity observed in pairwise comparison. Therefore, we confirmed the present species new to science and designated it as Myxobilatus channi n. sp. Histologically, plasmodia bulging toward the mucosa were confined to the circular muscle layer, leading to epithelial sloughing and necrosis of the intestinal villi. Phylogenetically, M. channi n. sp. was positioned branching from those species belonging to the family Myxobilatidae, implicating the inability of morphological taxonomy to reflect the true evolutionary relationship among myxosporeans. This study enhances the understanding of the composition of the parasite infecting the northern snakehead that will inform future scrutiny and management of the parasite infection.

Epididymis of the Lizard <i>Eutropis carinata</i>: A Light Microscopic and Ultrastructural Seasonal Study

The epididymis of the lizard Eutropis carinata can be divided into four regions viz., an initial segment (extra testicular) Efferent ductules (Eds) and three regions, the anterior region, a broad middle, and a narrow posterior, comparable to the caput, corpus, and cauda epididymidis of mammals. The anterior region of the epididymis is closely associated with a whitish initial segment (extra testicular), the Efferent ductules (Eds). The epididymal wall consists of an epithelial layer lining the lumen resting on a basement membrane surrounded by four to five layers of smooth circular muscles, a layer of connective tissue, and a layer of serosa. The ultrastructure study of the initial segment of epididymis the Eds during breeding season discloses four cell types in its epithelium viz., ciliated, non-ciliated, Apical-Mitochondria Rich Cells (AMRC), and basal cells. The anterior, middle, and posterior regions of the epididymis show five different cell types namely principal, basal, AMRC, narrow, and clear cells. Ciliated and non-ciliated cells are limited to the initial segment of the epididymis while, principal, narrow, and clear cells are found in the rest of the three regions of the epididymis. Basal and AMRC are found in all four regions. AMRCs are the most abundant cell type in the initial segment while principal cells are the major components of the epithelial lining of the remaining regions of the epididymis. During the non-breeding season, all the cell types are present but with regression and altered cytology of the cells without any sign of cellular activity in the different regions of the epididymis. During the breeding season, even the circulating testosterone levels are significantly higher compared to the non-breeding season. This is the first report describing different cell types in the initial segment and three different regions of the epididymis with ultrastructural seasonal variations in the Keeled Indian Mabuya, Eutropis carinata (Scincidae).

Substance P expression and neurochemical characterization in the enteric nervous system of the porcine colon

The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p<0.01-p<0.0001) with highest density in ISP of AC (p<0.0001 vs. DC). SP-IR neurons represent 27-28% of HuCD-IR neurons in ISP followed by OSP (19-22%) and MP (13-17%, p<0.05- p<0.0001) in AC and DC. The highest proportion of SP-IR neurons contains ChAT-IR (62-67% in the MP, 74-76% in the ISP, 70-85% in OSP) with no significant differences in the AC and DC. By contrast, SP-IR neurons containing nNOS-IR were much less abundant in both AC and DC with the highest peak in the OSP 36% vs. 16% in the ISP, p<0.01, and 20% in the MP in AC, whereas in the DC the highest peak was in the ISP (31%) followed by OSP and MP (25% and 18%) without significant differences. There was a small population of SP-/ChAT-/nNOS-IR enteric neurons in all plexuses in AC and DC. Varicose SP-IR fibers were abundant in the ganglia and distributed to the muscle layers and mucosa. Conclusions: SP-IR neurons include excitatory, inhibitory and interneurons based on the co-expression of ChAT, nNOS or both. The different density and neurochemical profile likely reflect different functions of SP-IR neurons such as immune modulation, regulation of mucosal function and control of vascular, myogenic and neurogenic activities. Mapping neuromodulators expression in a pre-clinical model provides the basis for elucidating neuronal circuits underlying GI functions and advance our understanding of human diseases. Supported by NIH SPARC OT2OD24899 & NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The substantia nigra modulates proximal colon tone and motility in a vagally-dependent manner in the rat

Previous studies have shown that a monosynaptic pathway connects the substantia nigra pars compacta (SNpc) to neurons of the dorsal motor nucleus of the vagus (DMV). This monosynaptic pathway modulates the vagal control of gastric motility. The present study was designed to test the hypothesis that this nigro-vagal pathway also modulates the tone and motility of the proximal colon.In rats, concurrent microinjection of a retrograde tracer in the proximal colon and an anterograde tracer in the SNpc (n=4) showed that bilaterally labelled colonic-projecting preganglionic vagal motoneurons in the DMV received inputs from SNpc neurons. Activation of SNpc neurons via microinjection of the ionotropic glutamate selective agonist, NMDA, increased proximal colonic motility (187±52.6% of baseline, n=13) and tone (323±97.2mg, n=14) as measured via a strain gauge aligned with the colonic circular smooth muscle (paired t test, p<0.0001). Following transfection of SNpc neurons with pAAV-hSyn-hM3D(Gq)-mCherry, chemogenetic activation of nigro-vagal nerve terminals by brainstem application of CNO increased proximal colon motility (157±24.5% of baseline; n=3; unpaired t test; p=0.0399). The increase in colon motility following SNpc neuronal activation was abolished by brainstem pretreatment with the dopaminergic DA1-like antagonist SCH23390 (n=4; one-way ANOVA, p=0.9762 vs baseline; p=0.0159 vs NMDA alone) as well as by acute subdiaphragmatic vagotomy (94±47.9% of baseline; n=3; One-way ANOVA, p=0.9816 vs baseline and p=0.0105 vs NMDA alone). In contrast to the excitatory effects observed upon activation of brainstem D1R following SNpc stimulation, DMV microinjection of DA decreased both the tone (-81±27.3mg, n=7) and motility (49±21.6% of baseline; n=7; paired t test: p=0.0004) of the proximal colon. These inhibitory DA effects were attenuated by application of the DA2-like antagonist L741646 (tone -100±58.9mg vs 99±100.1mg; n=4; paired t test: t3=2.567, p=0.0414 and motility 63±15% vs 144±38% of baseline; n=5; one-way ANOVA, p=0462 in the absence and presence of L741646).These data suggest that a monosynaptic nigro-vagal pathway activates brainstem D1 receptors to increase proximal colon tone and motility in a vagally-dependent manner. This study provides the anatomical and mechanistic basis to explain how loss of SNpc neurons may lead to gastrointestinal dysfunction, including constipation, observed in patients with Parkinson’s Disease. no disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of the aganglionic colon following surgical rescue in a cell therapy model of Hirschsprung disease in rat.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

Anaesthetic management and associated complications of per-oral endoscopic myotomy (POEM) procedure in a patient with achalasia cardia type II: A case report

Achalasia, a benign motility disorder of the esophagus, results in incomplete relaxation of the lower esophageal sphincter (LES) and absent peristalsis. Patients experience dysphagia, regurgitation, chest pain, weight loss, and heartburn. Pharmacological therapy has been unsatisfactory and definitive treatment has focused on mechanical disruption of the tight LES. Peroral endoscopic myotomy (POEM) is a safe and minimally invasive modality regarded as the first line management of all types of achalasia. POEM is performed under general anesthesia with endotracheal intubation using an orally inserted gastrointestinal endoscope. Briefly, POEM involves endoscopic creation of a mid-esophageal submucosal incision, creation of a submucosal tunnel with the endoscope, and then a myotomy of distal circular muscles with a Triangular Tip electrosurgical (TT) knife, resulting in relaxation of the achalasia. We, hereby report a case of a patient with Achalasia Cardia Type II posted for POEM procedure under General Anaesthesia.